Inhalation of asbestos fibers causes diffuse fibrosis of the lungs (asbestosis), pleural plaques and effusions, and an increased risk of cancer. Asbestos fibers have been shown to be acutely toxic to alveolar macrophages, pulmonary parenchymal cells, and mesothelial cells. The objective of this research is to identify the biochemical mechanisms responsible for acute asbestos toxicity. Two model systems were developed to reproduce the acute toxic effects of asbestos fibers in vitro and in vivo. The interaction between macrophages and crocidolite asbestos fibers generates reactive oxygen metabolites, including the highly reactive hydroxyl radical. Asbestos toxicity in vitro or in vivo is prevented by free radical scavenging enzymes or the iron chelator, deferoxamine. It is hypothesized that oxidant-induced stress directly or indirectly disrupts intracellular calcium homeostasis in macrophages exposed to asbestos fibers. The specific aims of the proposed research will evaluate whether the following biochemical mechanisms are responsible for altered calcium homeostasis and irreversible injury in these model systems. The first mechanism to be tested is production of DNA strand breaks by hydroxyl radicals or lipid peroxides leading to activation of poly (ADP-ribose) polymerase, depletion of adenine nucleotides, and cell death. The second mechanism to be tested is oxidation or depletion of cellular glutathione leading to release of calcium form the endoplasmic reticulum and cell death. The third mechanism to be tested is oxidation and hydrolysis of pyridine nucleotides leading to release of calcium from the mitochondria and cell death. Each of these biochemical changes will be measured directly in macrophages exposed to asbestos fibers in vitro and correlated with the onset of irreversible injury. Fluorescent probes will be used to monitor cell viability, free cytosolic calcium, and membrane-bound calcium on a single cell basis. These biochemical changes will be reproduced using specific metabolic inhibitors in order to assess whether these changes, alone or in combination, are sufficient to produce irreversible injury in macrophages. This experimental approach may ultimately provide a strategy to prevent acute toxicity and the chronic complications resulting from exposure to asbestos fibers.